Functional bioactive recombinant acylation stimulating protein is distinct from C3a anaphylatoxin.
Acylation stimulating protein (ASP) acts upon adipose tissue to stimulate triglyceride synthesis and glucose transport. The aim of the present study was to produce recombinant ASP and to measure its bioactivity. The cDNA region of the parent complement C3 sequence coding for ASP (C3adesArg) was cloned and expressed in E. coli. Bioactivity of the purified recombinant material was tested by determining its effect on triglyceride synthesis, glucose transport, and competition binding assays. In standard assays, concentrations of 5.5 microM recombinant ASP (rASP) stimulated triglyceride synthesis comparably to plasma ASP (pASP): 228% versus 237%, respectively, in 3T3 preadipocytes and 568% versus 440% in human differentiated adipocytes. rASP also increased glucose transport in L6 myocytes (163% at 10 microm rASP) and in human differentiated adipocytes (334% rASP vs. 329% pASP at 5 microM). rASP competitively displaced radiolabeled plasma ASP from high affinity association with the cell surface in both human differentiated adipocytes and 3T3 preadipocyte fibroblasts. Furthermore, immunoprecipitation of rASP and pASP with a specific monoclonal antibody abolished stimulation of cellular triglyceride synthesis. Lastly, we contrasted the structure:function activities of the arginated (C3a) and desarginated (ASP) proteins. The lipogenic activity and the anaphylatoxic activity result from distinct structural domains of the polypeptides. Thus rASP retains full biologic ASP activity and may provide a tool to study structure-function relationships in this physiologic system.
Murray
I
I
Mike Rosenbloom Laboratory for Cardiovascular Research, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada.
Parker
R A
RA
Kirchgessner
T G
TG
Tran
J
J
Zhang
Z J
ZJ
Westerlund
J
J
Cianflone
K
K
D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D001798:Blood Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002118:Calcium / Q000493:pharmacokinetics; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D015926:Complement C3a / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000494:pharmacology; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014280:Triglycerides / Q000096:biosynthesis
D001798:Blood Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014280:Triglycerides; C044541:complement C3a, des-Arg-(77)-; D015926:Complement C3a; D001120:Arginine; D005947:Glucose; D002118:Calcium